A number of electron dense molecules are used as contrasting agents for electron microscopy, including silver, lead, osmium, tungstates, ferritin, and colloidal gold particles. However, there are few very small ones that have site specificity. For example, labeling molecules with colloidal gold is adequate for many applications but is not precise enough to position the attachment site or control the stoichiometry of binding. Because colloidal gold attachment is noncovalent it is unstable under some conditions and because the binding to proteins is very pH dependent and requires low ionic strength, some proteins (such as IgM) are not easily labeled.
At the same time, a wide variety of antibody conjugates have been developed to be used in immunocytochemistry, in immunoassays, and in diagnostic imaging and therapy. For example, it has been shown that whole antibodies can be labeled with radioisotopes and used for the detection of cancer in humans (Burchiel et al., U.S. Pat. No. 4,311,688 and Carlsson et al., U.S. Pat. No. 4,232,119).
It has also been shown that the Fab portion of an immunoglobulin can be labeled and used in diagnostic and detection regimens [SJoquist et al., U.S. Pat. No. 3,966,898; Ling, U.S. Pat. No. 4,298,593; Aebi et al., PNAS., USA, 74:5514 (1977)].
It has also been shown that metal chelates can be conjugated to monoclonal antibodies and used in diagnostic and therapeutic techniques (Gansow et al., U.S. Pat. Nos. 4,454,106 and 4,472,509).
It has also been shown that electron dense moieties such as ferritin [Singer, Nature, 183:1523 (1959)] or colloidal gold [De Mey et al., U.S. Pat. No. 4,446,238 and Faulk et al., Immunochemistry, 8:1081 (1971)] can be used as markers for visualization of antibody labels in the electron microscope.
It has also been shown that immunodiffusion assay techniques can be developed using antibody-metal compound conjugates in which the metal or metal compound has a particle size of at least 5 nm (Leuvering et el., U.S. Pat. No. 4,313,734).
In the present invention, many of the drawbacks encountered by using the conventional contrasting agents for electron microscopy are overcome by using the dervatized gold clusters. These clusters are prepared by synthesizing a well-defined organometallic compound containing enough gold atoms for visibility in the electron microscope. The organic moiety may be designed to covalently attach to various groups on proteins, lipids, carbohydrates or other molecules. By using a molecular compound, the size is well defined and homogeneous. By using covalent coupling, the attachment may be performed under mild conditions without the requirement of low ionic strength. Also the resultant covalent coupling is stable under a wide range of conditions.
Further, in the present invention the derivatized gold cluster is used to prepare antibody or antibody fragment conjugates useful in diagnostic and therapeutic applications. Antibodies or active antibody fragments are covalently combined with a stable derivatized gold cluster. These clusters have an inner core of a specified number of gold atoms, for example 6, 8, 9, 11, 13, 25, 46, 55, and 67, and a shell of derivatized alkyl or aryl phosphines, preferably triaryl or trialkyl phosphines, with triphenyl phosphines being the preferred triaryl compounds, and/or mixed aryl and alkyl phosphines. The cluster also has coordinating counterions. The cluster may contain radioactive gold atoms when such activity provides diagnostic or therapeutic capabilities. These antibody-gold cluster conjugates retain the antibody specificity and activity, and are useful in any electron microscopy study, diagnostic technique, antigen localization study or therapy that relies upon the delivery of an effective dose to a targeted site.
Although each of the above-noted prior art technologies are adequate for some uses, each suffers from a deficiency now overcome by the present invention. Some of the labels noted above are generally too large for submolecular mapping in electron microscope studies. For example, an antibody molecule is about 15 nm in diameter and a colloidal gold complex is about 12-35 nm in diameter or larger (U.S. Pat. No. 4,446,238 discloses a diameter range of 20-40 nm). A conjugated label of these components is therefore about 27-50 nm in diameter; this is too large for meaningful electron microscopy studies in many fields where electron microscopy could be of value. In contrast, the antibody-gold cluster conjugates of the present invention can possess dimensions of about 5.0 nm, ideal for microscopy studies.
Some of the references noted above describe radiolabeled antibodies that are deficient because they are unable to couple more than a few radioactive elements onto an antibody without destroying the antibody activity, thus limiting the radioactive dose that can be delivered to a specific site (for imaging, diagnosis, or therapy). It is an object of the present invention to utilize a cluster system, whereby the antibody conjugate will contain multiple radionuclides, to increase the radiation dose to the targeted site. This application is of critical importance in the use of radiation to tumor therapy for certain forms of cancer. It is advantageous to deliver more radioactive dose per antibody so that the complete tumor mass receives an adequate dose. The derivatized radioactive gold cluster of the present invention can be covalently attached to specific antibody or antibody fragment sites such that the antibody or fragment carries a large number of radioactive gold atoms without significant loss of native immunospecificity. The 3.14 day half-life, 295 keV, and 66% beta emission (average range of 100 .mu.m) of the radioactive gold (.sup.199 Au) conjugates herein described are compatible with the use of these antibody-gold cluster conjugates in cancer therapy because these properties avoid the need for extended doses of radiation to the patient while still providing an effective cytotoxic dose.
It is also an object to take advantage of the small size of the antibody-gold cluster conjugates of the present invention. For example, it is possible to more effectively and efficiently treat or detect cancer by using the advantages inherent in the small size of, for example, a Fab'-Au.sub.55 conjugate, which permits penetration of tissues and tumors much more rapidly than previous attempts with whole antibodies coupled to radioisotopes. The small size of the conjugate also greatly improves resolution in electron microscopy studies in which specific sites or molecules or biological structures are labeled.
It is also an object of the present invention to provide a derivatized gold cluster containing enough gold atoms to be visible in the standard conventional electron microscope (CEM) and scanning electron microscope (SEM). The preferred cluster for such purposes containg 55 gold atoms. Image enhancement, using for example a silver developer, also permits the gold-55 clusters to be seen with conventional light miscroscopy for detection of cell types, tumors, viruses, and the llke.
It is also an object of the present invention to provide a cluster-conjugate in which several gold clusters may be coupled to an antibody or antibody fragment. Multiple detectable atoms via a cluster enables improved signal-to-noise ratios, and therefore earlier detection of small tumors.